Steroid, thyroid and retinoid hormones produce a diverse array of physiologic effects through the regulation of gene expression. Upon entering the cell, these hormones bind to a unique group of intracellular nuclear receptors which have been characterized as ligand-dependent transcription factors. This complex then moves into the nucleus where the receptor and its cognate ligand interact with the transcription pre-initiation complex affecting its stability and ultimately the rate of transcription of the target genes. The interactions of the liganded receptor with the specific elements in the promoter region are mediated by two classes of molecules; corepressors, which inhibit transactivation and coactivators, which enhance transactivation (Shibata et al., Recent Prog. Horm. Res., 1997, 52, 141-164).
ARA70 (also known as androgen receptor (AR) activator, ELE1, RET/PCT3, RFG for RET-fused gene and NCOA4 for nuclear receptor coactivator 4) is a member of the growing family of transcriptional coactivators. ARA70 was first isolated as a coactivator for the androgen receptor (AR) in human prostate cancer cells and has since been shown to act as a ligand-enhanced coactivator for the peroxisome proliferator-activated receptor gamma, another nuclear hormone receptor, in these same cells (Heinlein et al., J. Biol. Chem., 1999, 274, 16147-16152; Yeh and Chang, Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 5517-5521). ARA70 shows a broad tissue distribution of expression, including adipose tissue (Yeh and Chang, Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 5517-5521) and the DNA and protein sequence are disclosed in the PCT publication WO 97/44490 (Chang and Yeh, 1997).
Studies of androgen receptor regulation in prostate cancer cell lines have revealed a pivotal role for ARA70 in sex hormone signaling. By properly interacting with the androgen receptor, it has been suggested that ARA70 confers specificity of action between androgen and estrogen in the prostate (Miyamoto et al., Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 11083-11088; Miyamoto et al., Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 7379-7384; Yeh et al., Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 5527-5532).
Furthermore, cotransfection of ARA70 with the retinoblastoma protein (Rb), a tumor suppressor, in the prostate cancer cell line DU 145 has been shown to additively induce androgen receptor transcriptional activity by 13-fold with a concomitant reduction of cell growth (Yeh et al., Biochem. Biophys. Res. Commun., 1998, 248, 361-367).
ARA70 has also been identified in thyroid cancer cells, where chromosomal rearrangement resulted in the fusion of the genomic region of the RET tyrosine kinase to the 5' terminal region of the ELE1 gene. The resulting protein was designated in this context as RET/PTC3 or RET-fused gene (Bongarzone et al., Genomics, 1997, 42, 252-259; Minoletti et al., Genes Chromosomes Cancer, 1994, 11, 51-57; Santoro et al., Oncogene, 1994, 9, 509-516). More recently, it has been discovered that there are two isoforms of the ELE1 gene to which the RET tyrosine kinase can be fused to produce ARA70. The expression of these isoforms, designated alpha and beta (beta being a deletion variant most likely caused by the removal of one or more exons), thereby produce two isoforms of the ARA70 coactivator (Alen et al., Mol. Endocrinol., 1999, 13, 117-128).
Given the suggested role and expression pattern of ARA70 in prostate and thyroid carcinomas, the pharmacological modulation of ARA70 activity and/or expression may be an appropriate point of therapeutic intervention in pathological conditions.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of ARA70. Consequently, there remains a long felt need for agents capable of effectively inhibiting ARA70 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of ARA70 expression.
The present invention provides compositions and methods for modulating ARA70 expression, including modulation of the expression of the extended mRNA form of ARA70, known as RET-fused gene (RFG).